Cell growth matrix

ABSTRACT

The invention provides a structured cell growth matrix or assembly comprising a one or more spacer layers and one or more cell immobilization layers. The invention further provides a bioreactor comprising said matrix or assembly.

This application is a continuation of U.S. application Ser. No.16/933,398 filed on Jul. 20, 2020, which is a continuation of U.S.application Ser. No. 15/938,800 filed on Mar. 28, 2018, which is acontinuation of PCT/EP2017/078775 filed on Nov. 9, 2017, which claimspriority to Belgium application 13E2016/5839 filed on Nov. 9, 2016, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention concerns cell growth matrix, in particular astructured, high density cell growth matrix. The invention furtherconcerns the use of the matrix for growing cells and a bioreactorcomprising said matrix.

BACKGROUND

The techniques to culture cells such as eukaryotic cells, animal cells,mammalian cells and/or tissue are difficult and complex since thesecells are delicate and have nutrient and oxygen requirements duringgrowth which are complex and difficult to maintain. Given the increasingneed to culture cells in large quantities, bioreactors and culturingdevices have become an important tool in research and in the productionof cells for producing active proteins and/or antibodies and/or any cellby-products.

The bioreactors of the prior art comprise non-structured cell growthmatrixes and are provided with internal or external circulationmechanisms for cell culture medium circulation. The cell growth matrixesgenerally comprise carriers which can have the form of beads withregular or irregular structure, or may comprise woven or non-wovenmicrofibers of a polymer or any other material compatible with cellgrowth. The carriers can have a variety of forms and dimensions.

Several drawbacks can be attributed to the known bioreactors and theirnon-structured cell growth matrixes. The reproducibility lack ofvolumetric homogeneity of growth matrixes during their packing, andtheir movement and redistribution over time under the influence of theculture medium flow can lead to unreproducible cell culture environmentand very different micro-environments in different portions of thebioreactor. The carriers might gather in a given area inside thebioreactor thereby considerably lowering homogeneity during cellculture. Consequently, part of the cultured cells will have very limitedor no access to the culture medium and/or oxygen supply, which resultsin different cells metabolism, differences in product quality, even thedeath of said cells and a low cells production rate. Additionally, thenon-homogenous matrixes are difficult to produce and to pack given thevariable sizes and shapes of the carriers which might stick to eachother. Another drawback of the cell growth matrixes of the prior art isthat their cell culture surface cannot be determined with precision.Lastly, packing the carriers in the bioreactor is laborious and thuscostly.

U.S. Pat. No. 3,948,732 describes an assembly which includes aspiral-wound tubular chamber unit through which culture media and cellstravel within the chamber and about the center of the spiral and adhereto the inside wall of the chamber. The assembly includes a spacer memberprovided with spaced projecting support members which is interleavedwith the tubular chamber unit. These projecting members are intended tofacilitate gas flow through the cross section of the spiral assembly inan axial direction. At no time does the spacer member come into contactwith any of the cells or cell culture medium within the tube much lessprovide a path for the cells in any way.

It is the aim of the present invention to provide a cell growth matrixand a bioreactor comprising said matrix which overcome at least part ofthe above mentioned drawbacks. By preference, the cell growth matrixshould provide for a large cell growth surface within a small volumewhile still allowing circulation of medium and cells. Pressure dropswithin the system should be avoided as this is counterproductive tocertain cell viability. Reproducibility and homogeneity should beenhanced while maintaining manual operation and cost at an absoluteminimum. A tortuous path for cells and cell culture media to travel isneeded in conjunction with a spacer layer that facilitates that pathalong and parallel to the spacer layer and cell immobilization layers.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a cell growth matrixassembly or structured cell growth matrix.

In a second aspect, the present invention provides for the use of thecell growth matrix assembly or matrix according to any embodiment of theinvention for growing cells.

In a third aspect, the present invention provides a bioreactorcomprising a cell growth matrix assembly or matrix according to anyembodiment of the invention.

The cell growth matrix assembly or matrix of the current inventionpresent several advantages compared to those known in the prior art. Bymaking use of a structured assembly, variability during the cell growthprocess is omitted or minimalized. The cell culture surface can beeasily and accurately determined thanks to the known cell culturesurface and the number of the immobilization layers. The cell culturesurface to be placed inside a bioreactor can also be easily adaptedwithout modification of the matrix and/or the bioreactor design.

The matrix provides improved and organized cell and cell culture mediumflow inside a bioreactor.

Furthermore, the structured cell growth matrix provides efficientpacking inside a bioreactor thereby optimizing the use of the innerspace and increasing the cell growth surface inside said bioreactor. Thematrix thereby provides high cell density culture surface. Moreover,thanks to the structured design of the matrix, consistent andreproducible production of bioreactors containing said matrix isfacilitated.

DESCRIPTION OF FIGURES

FIGS. 1A and 1B show cross sectional views of portions of a matrixassembly according to two embodiments of the current invention.

FIG. 2A to D shows non-limitative examples of a matrix assemblyaccording to embodiments of the current invention.

FIG. 3A to B show a perspective and a top view of a matrix assemblypartially rolled according to an embodiment of the invention; and anembodiment of an assembly in tightly packed configuration.

FIG. 3C shows a top view of a matrix fully rolled, according to anembodiment of the current invention.

FIGS. 4A to 4D show examples of mesh spacer layer structures accordingto an embodiment of the current invention.

FIG. 5 shows a bioreactor chamber provided with a matrix assemblyaccording to an embodiment of the current invention, in which fluid andcells flow axially along the surfaces of the spacer layers andimmobilization layers between the top and bottom or bottom and top.

FIG. 6 shows a side view of a rotating bioreactor provided with a matrixassembly according to an embodiment of the current invention, rotatingaround its axis when in use.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a cell growth matrix assembly, the use ofthe latter for cell growth and a bioreactor comprising said matrixassembly.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

As used herein, the following terms have the following meanings:

“A”, “an”, and “the” as used herein refers to both singular and pluralreferents unless the context clearly dictates otherwise. By way ofexample, “a compartment” refers to one or more than one compartment.

“About” as used herein referring to a measurable value such as aparameter, an amount, a temporal duration, and the like, is meant toencompass variations of +/−20% or less, preferably +/−10% or less, morepreferably +/−5% or less, even more preferably +/−1% or less, and stillmore preferably +/−0.1% or less of and from the specified value, in sofar such variations are appropriate to perform in the disclosedinvention. However, it is to be understood that the value to which themodifier “about” refers is itself also specifically disclosed.

“Comprise,” “comprising,” and “comprises” and “comprised of” as usedherein are synonymous with “include”, “including”, “includes” or“contain”, “containing”, “contains” and are inclusive or open-endedterms that specifies the presence of what follows e.g. component and donot exclude or preclude the presence of additional, non-recitedcomponents, features, element, members, steps, known in the art ordisclosed therein.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within that range, as well as the recited endpoints.

The terms assembly, matrix assembly and matrix are used interchangeablethroughout the text.

In a first aspect, the current invention provides for a cell growthmatrix assembly, which comprises one or more cell immobilization layershaving a surface which allows cells to adhere and grow upon and forminga cell immobilization section. Adjacent to these cell immobilizationlayers are one or more spacer layers, including a structure which formsa spacer section allowing passage of cells and medium through an openbut tortuous path whereby the structure or nature of the spacer layersis chosen such that the spacer layers create a tortuous, open path forcells and culture media to travel in parallel to the surface of saidspacer and immobilization layers. The tortuous path or channel formed bythe spacer section creates turbulence which facilitates cell and cellmedium incursion into the immobilization layers.

Said cell immobilization layers will define a cell immobilizationsection, whereas said spacer layers define a spacer section.

In further or alternative embodiment, the present invention provides astructured cell growth matrix comprising one or more spacer sections andone or more cell immobilization sections. Each spacer section comprisesat least one spacer layer and each cell immobilization section comprisesat least one cell immobilization layer, wherein the thickness ratio ofthe immobilization section to the spacer section is at least 0.1 and thethickness of the spacer section is at least 0.1 mm. In an embodiment,the spacer section includes a structure that provides a tortuous path orchannel for cells to travel along the layers.

For the purpose of the current invention, a tortuous path is to beunderstood as a path with directional components that vary from andalong the general path.

The matrix assembly is designed such that it allows fluid and cells toflow axially along the surfaces of the spacer layer and cellimmobilization layer in a stationary bioreactor, or in the case of arotating bioreactor, tangentially along the surfaces of both layers. Thestructure of the spacer layers should be thus chosen that it creates atortuous, yet open path in between immobilization layers, therebycreating turbulence. This turbulence will drive cells and medium intothe immobilization layers.

It will be understood by the skilled person that such tortuous pathspacer could be achieved by multiple ways, all readily known in the art.In one embodiment, the spacer layer is comprised of a bearing structureor spherical, near-spherical or egg-shaped objects such as beads, packedas a three-dimensional structure on top of each other (see FIG. 1B). Abearing structure is to be understood as a structure formed of balls orspheres optionally fixed to a surface support, which form a tortuouspath through which fluid may flow.

In another embodiment, the spacer layer may be a mesh or comprises amesh structure. For the purpose of the current invention, mesh structureor mesh is to be understood as a structure comprising a network orweb-like pattern of filament, wire or thread, said network definespores, openings or perforations formed of a three dimensional weave.Examples of mesh structures are given in FIGS. 4A to 4D. It will beunderstood that these cannot be seen as limitative to the currentinvention.

The cell growth matrix assembly or matrix according to the currentinvention provides for a substrate that allows high density cell growth.By preference, a high cell density bioreactor allows for the maximum ofcell growth surface in a minimum of volume. The design of the currentcell growth matrix assembly is optimized to meet these demands. Byproviding a spacer section adjacent to the immobilization section, celland medium flow is ensured. These spacer sections promote turbulence andallow essentially tangential flow in between cell adherence sections, inaddition to random perpendicular flow through the immobilization layers.This increases cells adherence to the immobilization layers of theimmobilization sections.

Additionally, the cells are more homogenously distributed inside thebioreactor thanks to the spacer sections which provide space for cellmovement until adherence, and which prevents the immobilization sectionsfrom acting as filters. The spacer further allows improved removal oftoxic metabolites by providing space for the movement of saidmetabolites.

The spacer section further allows better distribution of the culturemedium and thereby of the nutrients inside the bioreactor. All cellsinside the bioreactor are equally provided with culture medium. Giventhe structured design of the matrix, preferential culture medium routewill not be created inside a bioreactor. This is an improvement over thenon-structured matrixes of the prior art, which generate preferentialculture medium routes when used in a bioreactor as the non-structuredcarriers move under the influence of the culture medium flow. The resultis a non-homogeneous cell distribution.

In order to ensure a minimal of pressure drop and volume loss, thespacer section is comprised of a structure including a tortuous path forcell and fluid flow. In one embodiment, the structure is a mesh. Byusing a mesh structure for spacer section, the thickness of the physicalbarrier is kept to an absolute minimum thereby ensuring constantpressure and yet still allowing sufficient space between the neighboringimmobilization sections.

In an embodiment the structure or mesh size of the spacer layer will bebetween 0.05 mm to 5 mm. The choice of the structure or mesh size isimportant as it again influences the balance between providing asufficient barrier between the immobilization sections thereby allowingcell and medium passage, whilst ensuring adequate pressure in the systemand achieving a high cell density. The openings in the mesh or grid canbe of any shape, such as round, elliptical, square, or rectangular. Inan embodiment, the size of the openings is at least 0.05 mm, at least0.06 mm, at least 0.08 mm, at least 0.1 mm, at least 0.15 mm, at least0.2 mm or at least 0.25 mm. In another or further embodiment, the sizeof the openings is at most 5 mm, at most 4.5 mm, at most 4 mm, at most3.5 mm, at most 3 mm or any value comprised in between theaforementioned values.

It will be understood that the ratio of the spacer section, which isbuilt from one or more spacer layers will have an impact on thefunctioning of the matrix assembly. In an embodiment the thickness ofthe spacer section is at least 0.1 mm, more preferably between 0.1 mmand 5 mm, more preferably between 0.2 mm and 1 mm. In an embodiment, thethickness of the spacer section is between 0.25 and 0.6 mm, such as 0.4mm or 0.5 mm.

In an embodiment the thickness in mm of the spacer section is at least0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1,at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, atleast 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, atleast 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, atleast 3.2, at least 3.4, at least 3.5, at least 3.6, at least 3.7, atleast 3.8, at least 3.9, at least 4, at least 4.1, at least 4.2, atleast 4.3, at least 4.4, at least 4.5, at least 4.6, at least 4.7, atleast 4.8, at least 4.9, at least 5, at least 5.5, at least 6, at least6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, atleast 9.5, at least 10 or any value comprised between the aforementionedvalues.

In a further or alternative embodiment, the thickness in mm of thespacer section is at most 1000, at most 950, at most 900, at most 850,at most 800, at most 750, at most 700, at most 650, at most 600, at most550, at most 500, at most 450, at most 400, at most 350, at most 300, atmost 250, at most 200, at most 190, at most 180, at most 170, at most160, at most 150, at most 140, at most 130, at most 120, at most 110, atmost 100, at most 95, at most 90, at most 85, at most 80, at most 75, atmost 70, at most 65, at most 60, at most 55, at most 50, at most 45, atmost 40, at most 35, at most 30 at most 25, at most 20, at most 15, atmost 12 or any value comprised between the aforementioned values.

The thickness of the cell immobilization section will equally be ofimportance to the functioning of the system. A cell immobilizationsection which is too thick will result in a poorly populated area,whereas a section which is too thin will have a negative impact on theavailable cell growth surface, again negatively influencing cell growth.By preference, the thickness of the immobilization section will bebetween 0.1 mm and 15 mm, more preferably between 0.1 mm and 10 mm, evenmore preferably between 0.1 and 5 mm, or between 0.1 mm and 1 mm.

There is a need for a balance between sufficient cell and medium flowbetween the cell growth surfaces and a sufficiently large cell surfacefor cell growth. The inventors of the current invention have found thatideally, the ratio between the thickness of the cell immobilizationsection to the spacer section should be at least 0.1, and morepreferably between 0.1 and 5, even more preferably between 0.1 and 2,such as e.g. 1:1. As such, the needs of the system are met.

In an embodiment, the thickness ratio of the immobilization section tothe spacer section is at least 0.1, at least 0.2, at least 0.3, at least0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4,at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, atleast 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, atleast 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, atleast 3, at least 4, at least, 5, at least 6, at least 7, at least 8, atleast 9, at least 10 or any value comprised between the aforementionedvalues. In a further or alternative embodiment, the thickness ratio ofthe immobilization section to the spacer section is at most 50, at most45, at most 40, at most 35, at most 30, at most 25, at most 20, at most19, at most 18, at most 17, at most 16, at most 15, at most 14, at most13, at most 12, at most 11 or any value comprised between theaforementioned values.

The spacer layers and/or the cell immobilization layers which make uprespectively the spacer section and the immobilization section in thematrix assembly are preferably made of a biocompatible polymer selectedfrom polyester, polyethylene, polypropylene, polyamide, plasma treatedpolyethylene, plasma treated polyester, plasma treated polypropylene orplasma treated polyamide. Said layers can be hydrophilic or hydrophobic.The cell immobilization layers are preferably hydrophilic.

The thickness of both layers will advantageously be between 0.05 mm and3 mm, more preferably between 0.1 and 2 mm or between 0.1 and 1 mm.

Suitable material for the cell immobilization layer may be a woven ornonwoven material. By preference, a nonwoven material is used. Anonwoven, contrary to a woven material, is a fabric which is not createdby weaving or knitting and does not require converting the fibers toyarn. Nonwovens are broadly defined as sheet or web structures bondedtogether by entangling fiber or filaments (and by perforating films)mechanically, thermally or chemically. The nature of the nonwovenmaterial used in the current application may be of any origin, eithercomprising of natural fibers or synthetic fibers. By preference, thenonwoven is made of a polymer, such as polyester or polypropylene. Thecell immobilization layers used in the current invention may be chosenfrom a polyethyleentereftalate nonwoven. The nonwoven material may beplasma treated to enhance cell adherence and flow.

The spacer layers may consist of a (biocompatible) polymer with meshsize as described above. In one embodiment, the spacer layer is asynthetic woven fabric or structure. In another embodiment, the spacerlayer is a bearing structure. Such structure may be produced from abiopolymer (e.g. alginate). Other suitable material for this purpose issilica, polystyrene, agarose, styrene divinylbenzene, polyacrylonitrileor latex.

The spacer layer may be gamma irradiated in order to reduce bioburden.

The design of the matrix assembly can take many forms depending on theapplication and type of bioreactor.

In an embodiment of the current invention, the immobilization sectionand spacer section are alternately positioned. Alternately positionedmeans that each spacer section is followed by a cell immobilizationsection which is itself followed by a spacer section. The alternatelypositioned sections may alternate in vertical position as shown in thefigures (see further) or in a horizontal position according to the useof the matrix and/or to the bioreactor in which the matrix will beintroduced.

In this embodiment, one or more layers of cell immobilization layers aresuperimposed on one or more spacer layers (or vice versa). Thisconfiguration may be repeated several times if deemed required in ordercreating a stack of several immobilization and spacer sections. Ideally,the end configuration may comprise between 1 and 500 alternations is ofthe above described layering. The stacked layers may be positioned in aframe or cassette or sealed/connected at their circumference. In anotherembodiment, the achieved stack can be rolled around an axis or core toachieve a spiral configuration.

The amount of layers used in both the immobilization section and spacersection can be chosen based on the application, characteristics of thelayers (dimensions, size, etc.)

and desired result. Hence, the amount of layers within eitherimmobilization section or spacer section may be between 1 and 20, morepreferably between 1 and 10, even more preferably between 1 and 5.

As mentioned, the presence of the spacer sections creates space insidethe matrix through which the culture medium flows. This providesimproved circulation of the culture medium through the matrix therebyreaching all cultured cells. This effect is even more enhanced in theembodiment wherein the spacer section comprises one spacer layer and theimmobilization section comprises two immobilization layers. The culturemedium flowing inside the matrix via the spacer sections is tangentiallyoriented with respect to the cell immobilization sections.

The spacer sections improve the rigidity of the matrix thanks to therigidity of the spacer layers. The matrix according to any embodiment ofthe invention can be compressed by any method known to the personskilled in the art. The size of the compressed matrix is reduced bymaximum 20%, preferably maximum 15%, more preferably maximum 10%compared to the size of the non-compressed matrix.

In an embodiment, one surface of at least one spacer layer or section isat least partially free from coverage by any cell immobilization layersor other layer or section. The non-covered layer section or layer isdesigned to be positioned in contact with the inner wall of anybioreactor. This design allows preventing plunger effect inside thebioreactor.

In an alternative embodiment of the current invention, the matrixassembly is comprised of a configuration of one or more cellimmobilization layers as described above forming an immobilizationsection and one or more spacer layers as described above forming aspacer section, positioned adjacent to (e.g. above and/or under) saidcell immobilization layers. Optionally, the layering may be repeated toform an alternated, stacked configuration of one or more cellimmobilization layers and one or more spacer layers. Said resultingconfiguration is subsequently spiral- or concentrically wound along anaxis or core. The thickness of the spacer section may be between 0.1 mmand 5 mm, more preferably between 0.2 mm and 1 mm, whereas the thicknessof the immobilization section may be between 0.1 and 5 mm. The ratio ofthickness of the immobilization section over the thickness of the spacersection is preferably between 1:2 to 2:1, most preferably 1:1. While theamount of layers to achieve this may vary based on the characteristicsof the layers used and thus freely chosen as described above, it wasfound that a good result was achieved when two immobilization layers areused and one spacer layer. The thickness of the spacer andimmobilization layers is between 0.05 mm and 3 mm, more preferablybetween 0.1 and 2 mm or between 0.1 and 1 mm. While the material of theimmobilization and spacer layer should not be construed as limitative,the immobilization layer is preferably made from a woven or nonwovenfabric or material as described above for the other embodiments. Saidspacer layer may also be formed of a woven or nonwoven material and mayinclude a mesh structure.

By preference, the outer layer of the assembly, being the side facingthe wall of the bioreactor when placed within the bioreactor will be aspacer layer. This prevents cell growth against the wall of the reactor.Such bioreactors may be heated and cell growth in this area couldcompromise cell yield and/or quality. In a further embodiment, severalspacer layers are provided at the outer border of the assembly, again toensure good insulation. In another or further embodiment, an insulationlayer which is made of another material than the spacer layer isprovided.

The assembly of the matrix is simple and repeatable compared to those ofthe prior art. The invention further offers scalable matrixes byoffering the possibility of easily producing matrixes having specificdimensions and/or cells growth surface. The scalability of theassembly/matrix of the invention has no impact on the homogeneity and/orthe quality of the culture medium flow provided.

In another aspect, the present invention provides for the use of theassembly/matrix according to any embodiment described above for growingcells.

In another aspect, the present invention provides a bioreactorcomprising an assembly or matrix according to any embodiment describedabove. The bioreactor might comprise more than one assembly or matrix.In a further embodiment, the assembly/matrix and/or the bioreactorcomprising said matrix are single use. The assembly/matrix can bepositioned vertically or horizontally in the bioreactor.

Preferably, the assembly/matrix represents at least 10%, at least 20%,at least 30%, at least 40% or at least 50% of the bioreactor innerspace. Said matrix represents at most 100%, at most 90%, at most 80%, atmost 70% or at most 60% of the bioreactor inner space.

The bioreactor can be any type of bioreactor known to the person skilledin the art such as perfusion bioreactor, wave bioreactor, cylindricalbioreactor, rotating bioreactor, bag bioreactor, moving bed bioreactor,packed bed bioreactor, fibrous bioreactor, membrane bioreactor, batchbioreactor, or continuous bioreactor. The bioreactor can be of any shapeand can be made from any material, for example, stainless steel, glass,or plastic.

It is supposed that the present invention is not restricted to any formof realization described previously and that some modifications can beadded to the presented example without reappraisal of the appendedclaims.

FIGURES

FIGS. 1A and 1B show cross-sectional views of portions of a matrixassembly according to two embodiments of the current invention. Byproviding one or more spacer layers 3 or 3′ between one or more cellimmobilization layers 2 (forming the immobilization section 10),turbulence (depicted with black arrows) and a random perpendicular flow(horizontal open arrows) is promoted. The assembly organises a fluidpath which allows sufficient flow distribution when used within a fixedbed. By this design, liquid flow homogeneity is ensured and as aconsequence, cells are equally homogenously distributed over theassembly. By providing a spacer creating a tortured, open path, cellsand medium flow is allowed along the surface of both layers. Theturbulence will push cells and medium deeper into the cellimmobilization layers. Overall, the design promotes reproducibility andhomogeneity when used for cell production.

FIG. 1A depicts an embodiment wherein the spacer section 11 is comprisedof one or more spacer layers 3 which are made of a mesh fabric. Examplesof possible mesh fabrics which can be used for this purpose are shown inFIGS. 4A to 4D, showing various forms of openings.

FIG. 1B depicts an alternative embodiment wherein the spacer section ismade of spherical or near-spherical objects such as beads.

Both the mesh structure and the beads are examples of structures orlayers which providing a tortured, open path as described above.

FIGS. 2A to 2D show possible arrangements of the number ofimmobilization layers versus spacer layers which respectively make upthe cell immobilizations section 10 and spacer section 11.

The thickness of each section corresponds to the sum of the thickness ofthe layers contained therein. In a one embodiment, the spacer section 11comprises one spacer layer and the cell immobilization section 10comprises two cell immobilization layers as shown in FIGS. 2A and 2B. Inthis configuration, at least one surface of each immobilization layer isin contact with one surface of the spacer layer. FIGS. 2C and 2D showalternative examples, wherein the spacer section 11 comprises one spacerlayer and the immobilization section comprises three immobilizationlayers.

The spacer sections and the cell immobilization section might be of anyshape and might have similar of different dimensions. The matrix can beformed by alternating the different sections thereby obtaining a threedimension (3D) matrix. Said matrix can have any geometrical shape suchcylindrical, triangular, rectangular or any irregular 3D shape. Thematrix can also have other shapes obtained by further shaping the 3Dmatrix such as rolling the 3D matrix to obtain a spiral.

An example of a rolled matrix is shown in FIGS. 3A to 3B showingrespectively a perspective view and a top view of the rolled matrix.These figures show a matrix or assembly which is “loosely” rolled toexemplify the spiral structure. It is to be understood that the degreeof rolling the matrix is variable according to the use of the assemblyand/or the wish of the user. The assembly or matrix will be tightlyrolled to a structure such as the spiral structure shown in FIG. 3C.

FIG. 3C shows an embodiment of the current invention, whereby one ormore cell immobilization layers 2 are adjacent to one or more spacerlayers 3 made from a mesh structure and whereby the layering mayoptionally be repeated several times to achieve a stacked configuration.Examples of mesh structures are given in FIGS. 4A to 4D (enlarged view).Preferably, the mesh structure included in spacer layers of the currentinvention forms a tortuous path for cells and fluid to flow when layeredbetween two immobilization layers. Alternatively, other spacerstructures can be used which form such tortuous paths. The embodimentshown in FIG. 3C is shown as subsequently spirally or concentricallyrolled along an axis or core whereby all layers are firmly wound. Thediameter of the core, the length and/or amount of the layers willultimately define the size of the assembly or matrix. By preference, thethickness of the layers will be between 0.1 and 5 mm. In the embodimentshown in FIG. 3D, the thickness of the layers is between 0.25 and 0.7mm, whereby two layers of cell immobilization layers are alternated byone spacer layer. By preference, the outer layer of the resulting spiralconfiguration will be one or more spacer layers. Alternatively, aninsulating layer could be provided as outer layer, which differs fromboth the spacer and cell immobilization layer. The spacer layers are bypreference a woven mesh fabric with mesh size between 0.05 mm and 5 mm.The cell immobilization layers are preferably a nonwoven fabric.

FIGS. 5 and 6 show respectively a stationary bioreactor and a rotatingbioreactor provided with a matrix assembly according to embodiments ofthe current invention. In FIGS. 5 and 6, the matrix as shown in FIG. 3Cis included in the bioreactor chambers. In both embodiments, the flow ofcells and medium will be along the path created by the spacer layers.

In the embodiment as shown in FIG. 5, culture media and cells flowthrough the matrix assembly in the axial direction along the surface ofthe spacer and immobilization layers (from top to bottom or bottom totop).

FIG. 6 shows a rotating bioreactor whereby the matrix assembly ispartially submerged in medium, and whereby the bioreactor and matrix arerotated along their axis. In this embodiment, the flow of medium andcells will be in the tangential direction along the surfaces of thespacer and immobilization layers (in the spiral path).

1. An apparatus for culturing cells, comprising: a bioreactor includinga matrix assembly for growing cells in connection with a cell mediumflowing through the matrix assembly, the matrix assembly comprising atleast two cell immobilization layers in direct contact.
 2. The apparatusof claim 1, the bioreactor having a fluid flow direction aligned with amajor dimension of the matrix assembly.
 3. The apparatus of claim 1,wherein the at least two cell immobilization layers comprise wovenlayers.
 4. The apparatus of claim 1, wherein the at least two cellimmobilization layers comprise woven mesh layers.
 5. The apparatus ofclaim 1, further including at least one non-woven layer.
 6. An apparatusfor culturing cells, comprising: a bioreactor including a matrixassembly for growing cells in connection with a cell medium flowingthrough the matrix assembly, the matrix assembly comprising at least onewoven mesh layer and at least one non-woven layer.
 7. The apparatus ofclaim 6, wherein the matrix assembly comprises a plurality of woven meshlayers.
 8. The apparatus of claim 7, wherein the plurality of woven meshlayers comprise at least a first woven mesh layer in direct contact withat least a second woven mesh layer.
 9. The apparatus of claim 6, whereinthe at least one non-woven layer is in contact with one of the first orsecond woven mesh layers.
 10. An apparatus for culturing cells,comprising: a bioreactor including a matrix assembly for growing cellsin connection with a cell medium flowing through the matrix assembly,the matrix assembly comprising at least one woven mesh layer.
 11. Theapparatus of claim 10, wherein the matrix assembly comprises a pluralityof woven mesh layers.
 12. The apparatus of claim 11, wherein theplurality of woven mesh layers comprise at least two woven mesh layersin direct contact.
 13. The apparatus of claim 12, wherein the pluralityof woven mesh layers are arranged in a stacked configuration.
 14. Theapparatus of claim 12, further including a plurality of cellimmobilization layers arranged in a stacked configuration with theplurality of woven mesh layers.
 15. The apparatus of claim 10, furtherincluding a plurality of cell immobilization layers each comprising anonwoven material.
 16. The apparatus of claim 10, wherein the bioreactorcomprises a roller bottle.
 17. The apparatus of claim 10, wherein thebioreactor is adapted for passing the cell medium in a vertical flowthrough the matrix assembly.
 18. The apparatus of claim 17, wherein thebioreactor is adapted to create the vertical flow through the matrixassembly from bottom to top.
 19. The apparatus of claim 17, wherein thebioreactor is adapted to create the vertical flow through the matrixassembly from top to bottom.
 20. The apparatus of claim 10, wherein thematrix assembly is spiral wound.
 21. The apparatus of claim 10, whereinthe matrix assembly is spiral wound about a core.
 22. The apparatus ofclaim 10, wherein the matrix assembly is annular.
 23. The apparatus ofclaim 10, wherein the at least one woven mesh layer comprises openingshaving a shape selected from the group consisting of round, elliptical,square, or rectangular.
 24. The apparatus of claim 10, wherein the atleast one woven mesh layer comprises openings having a size of betweenat least 0.05 mm and 5 mm.
 25. The apparatus of claim 10, the bioreactorhaving a fluid flow direction along the at least one woven mesh layer.26. The apparatus of claim 10, further including at least one non-wovenlayer.